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AAV Purification by Iodixanol Gradient Ultracentrifugation

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Introduction

This protocol can be used to purify AAV of any serotype.

In medicine, iodixanol can be used as an intravenous isomolar contrast agent. In research, iodixanol is used as an isomolar density gradient medium suitable for virus purification and isolation of cells, organelles, lipoproteins, and macromolecules. Importantly, iodixanol is inert and non-toxic to mammalian cells. Therefore, and unlike other density gradient media, it is reportedly not necessary to remove iodixanol prior to use of your purified virus. Nonetheless, we recommend performing a buffer exchange before using the purified AAV in vivo .

The iodixanol gradient in this protocol is composed of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol step has 1M NaCl to destabilize ionic interactions between macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids. The 60% step serves as a cushion for genome-containing virions. Phenol red is added to clearly distinguish the steps. This method will help enrich the prep with full (genome containing) particles. On average, iodixanol-purified AAV preparations contain ~20% empty particles.

60% iodixanol solution is available under the name
OptiPrep
from Sigma-Aldrich.

Last Upload: January 17, 2018

Protocol Video

Watch the protocol video below to learn setup and use an iodixanol column gradient for AAV purification.

Workflow Timeline

Day 1: Purify

Day 2: Buffer exchange and concentration

Note: Both steps could be completed in one (long) day.

Equipment

Ultracentrifuge

T70i rotor or equivalent

Reagents

Opti-Prep (60% Iodixanol)

QuickSeal tubes

QuickSeal tube spacers

16 ga needle

18 ga needle

10 mL syringe

1x PBS pH 7.4

1x PBS-MK buffer

100X Pluronic-F68

NaCl

MgCl2

KCl

Centrifugal filter units (MWCO 100 kDa)

Reagent Preparation

1 M NaCl/PBS-MK buffer

Dissolve 5.84 g of NaCl, 26.3 mg of MgCl2and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing through a 0.22-μm filter and store at 4 ℃.

1x PBS-MK buffer

Dissolve 26.3 mg of MgCl2, and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing through a 0.22-μm filter and store at 4 ℃.

0.001% Pluronic-F68 (formulation buffer)

Add 5 μL of 100X Pluronic F-68 to 49.95 mL of PBS. Store at 4 ℃ for up to one month, or aliquot and store at -80 ℃ for up to one year.

Overlay each solution into a QuickSeal tube in the order below using a 10 mL syringe and an 18 g needle, taking care to avoid bubbles (Figure 1).

5 mL of 60% iodixanol step

5 mL of 40% iodixanol step

6 mL of 25% iodixanol step

8 mL of 15% iodixanol step

Carefully add up to 5 mL of clarified supernatant on top of the gradient. Use 1x PBS (or cell lysis buffer) to top off the tube.

Seal the QuickSeal tubes.

Centrifuge at 350,000 g for 90 min in a T70i rotor at 10 ℃.

*Pro-Tip* If you need more time, you can alternatively centrifuge for 2 h at 200,000 g at 18 ℃.

Carefully take the QuickSeal tubes out of the rotor and place them in a stable rack. **Make sure not to disturb the gradient!**

Collect Fractions

Option #1

Prepare a row of roughly 20 open 1.5 mL microcentrifuge tubes in a rack. These will be used to collect fractions from the gradient. You can collect fewer fractions once you have a good idea when the virus elutes from the gradients.

In a biosafety cabinet, carefully puncture the QuickSeal tube at the interface of the 60% and 40% gradient (see Figure 2) with an 18 g needle.

Place the first microcentrifuge tube under the needle’s opening to collect the fractions.

Puncture the top of the QuickSeal tube with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per tube.

Avoid the proteinaceous material near the 40-25% interface.

*Pro-Tip* As soon as the top of the tube is punctured, AAV-containing Iodixanol solution will flow from the needle set at the 40-60% interface. Make sure that the microcentrifuge tubes are well positioned to collect the solution or you will lose a significant amount of your virus. When the first fraction is collected, move the rack to the next empty tube to collect the next fraction. The flow will be rapid at first and will progressively slow down.

Add more sample and spin 3500 rpm for 4 min at 4°C, discard the flow through. Repeat this step as needed.

Please note that iodixanol is not easily removed. After each spin, add more formulation buffer and sample and make sure to pipet back and forth a few times to mix the iodixanol that has settled at the bottom of the column.

We recommend concentrating to a minimum of 500 µl. If the concentrate volume is less than 500 µl, bring up the volume with formulation buffer.

Use a P1000 to the bottom of the filter and pipette up/down and wash off the walls of the filter to recover as much virus as possible.

Store at 4 ℃ for short term (2 weeks), or aliquot and store at -80 ℃ for long term.